| Adriamycin is a kind of antitumor antibiotics,mainly for acute and chronic leukemia and malignant lymphoma,breast cancer and other solid tumors.However,in the clinical application,the limitations of Adriamycin,which seriously affect the treatment effect of Adriamycin and the physical and mental health of patients.Therefore,it is necessary to find drugs that can reduce the cardiac toxicity of Adriamycin.Fucoxanthin also known as algin,is a kind of natural carotenoid.Many studies show that in recent years,Fucoxanthin has a strong antioxidant,antitumor activity.This study used Fucoxanthin and Adriamycin as raw materials,mainly by means of methods of molecular biology and cell biology,investigates the protective effect of Fucoxanthin on Adriamycin induced cardiotoxicity,Fucoxanthin and Adriamycin combination of human breast cancer cell line MCF-7 synergistic inhibitory effect.To provide a new way for the clinical application of adriamycin and the clinical treatment of breast cancer.Contents 1.This topic used Fucoxanthin and Adriamycin as experimental materials;the cultured rat primary myocardial H9c2 cells in vitro as the research object,from the following five aspects explore the protective effect of Fucoxanthin to Adriamycin induced cardiotoxicity:（1）The DPPH method used to determinate the free radical scavenging effect of Fucoxanthin and the Vitamin C as a control to determine the antioxidant capacity of Fucoxanthin.（2）The MTT assay was used to detect different concentrations of Adriamycin and combined with different concentrations of Fucoxanthin on H9C2 cells apoptosis rate,for detecting the protective effect of Fucoxanthin to H9C2 cells.（3）AO/EB staining to observe the morphological changes of H9C2 cells which treated with Adriamycin and combined with Fucoxanthin.（4）Determination of ROS level in H9C2 cells treated by combined with Fucoxanthin combined with Adriamycin in Carboxy-DCFDA assay.（5）Western blotting detected the expression of apoptosis protein in H9C2 cells which treated by Fucoxanthin combined with Adriamycin.2.This topic to Fucoxanthin and Adriamycin as experimental material,human breast cancer MCF-7 cells cultured in vitro as the research object,from the following four aspects to explore Fucoxanthin and Adriamycin in combination with inhibitory effect on tumor cells:（1）MCF-7 cells were treated with different concentrations of Fucoxanthin and Adriamycin,MTT assay to detect inhibition rate on MCF-7 cells and the two drugs combined index is calculated according to the median effect principle.（2）AO/EB staining to observe the morphological changes of MCF-7 cells which treated with Fucoxanthin and Adriamycin.（3）JC-1 kit to detect the combined effects of Fucoxanthin combined with Adriamycin on the mitochondrial membrane potential of MCF-7 cells.（4）Western blotting detected the expression of apoptosis protein in MCF-7 cells which treated by Fucoxanthin combined with Adriamycin.Results 1.The protective effect of fucoxanthin on adriamycin-induced cytotoxicity in cardiac muscle cells:（1）The free radical scavenging rate of Fucoxanthin and Vitamin C in concentration12.5μmol/L were 64.2%and 33.0%respectively,with IC50were 10.88μmol/L and14.37μmol/L.（2）Fucoxanthin combined with Adriamycin on H9C2 cells inhibition rate decreased significantly,when doxorubicin concentration of 2μmol/L,Fucoxanthin concentration is 0μmol/L on H9C2 cells inhibition rate was 52.75±4.98%.When Adriamycin concentration at 2μmol/L,Fucoxanthin concentrations at 50μmol/L inhibition rate of H9C2 cells was 37.80±2.57%.The inhibition rate reduced 12.95±2.41%（p<0.05）.（3）Fluorescence microscope:the blank control group and Fucoxanthin alone group had no obvious cell apoptosis.Adriamycin alone group appeared a large number of H9C2 early apoptotic cells,the expression of the nucleus for AO staining showed a yellow green fluorescence,concentrated in a crescent shaped or granular,located on the side of the cell.In addition,there are a few late apoptotic cells and apoptotic bodies appeared,which showed that the nucleus was orange red,concentrated and in favor of EB staining.The Fucoxanthin and Adriamycin induced apoptosis of H9C2 cells in ADR group was significantly lower than that of the single group number.（4）Normal group and Fucoxanthin single group of H9C2 cells and ROS level is low（p<0.05）and Adriamycin single group H9C2cells and ROS level was significantly higher than that of the normal group and the Fucoxanthin single group（P<0.01）,and Fucoxanthin and Adriamycin combined group H9C2 cells ROS levels compared with Adriamycin group significantly decreased but higher than that of the normal group and the Fucoxanthin single group（p<0.05）.（5）Fucoxanthin can inhibit the regulation of Adriamycin induced cardiomyocyte apoptosis protein Caspase-8 up and Cleaved Caspase-3,Cleaved PARP down in H9C2 cells.2.Fucoxanthin combined with adriamycin on promoting the apoptosis of breast cancer cell:（1）The inhibition rate to MCF-7 of Fucoxanthin single group,Adriamycin single group and combination group of cell were with the dose increased and increased.When the dosage of the two drugs is large,they were antagonistic.When the dosage of the two drugs is low,they were coordination.（2）MCF-7 cells by AO/EB staining results:blank control group,nuclear chromatin was green and showed normal structure;Fucoxanthin a single group of cells,nuclear chromatin,green showed pyknosis or bead like shape;Adriamycin monotherapy group cells,vacuoles,nuclear chromatin showed a strong yellow green fluorescent light;Fucoxanthin and Adriamycin combined cell group is in the form of late apoptosis,nuclear chromatin orange red and showed pyknosis or bead like shape.（3）The blank control group,cell mitochondrial membrane potential decreased the ratio of0.09±0.47%and Fucoxanthin single group cell mitochondrial membrane potential decreased the ratio of 2.40±1.43%,Adriamycin single group cell mitochondrial membrane potential decreased the ratio of 4.12±2.71%,whereas the coadministration group cell mitochondrial membrane potential decreased the proportion of 20.02±4.84%（p<0.05）.（4）Cleaved-Caspase-3 and PARP cleavage were significantly increased in the combined treatment group,but also caused the up regulation of Bax and the down-regulation of Bcl-2,and ultimately the apoptosis pathway of MCF-7.Conclusion 1.Fucoxanthin can protect the cardiotoxicity induced by Adriamycin,and its protective effect through its antioxidant activity inhibition to Adriamycin induced myocardial cell apoptosis.2.Fucoxanthin combined with Adriamycin had the inhibition of human breast cancer MCF-7 cells are cooperative.When they were combination,they had better antitumor effect. |
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